First identified by molecular cloning in 1989 (Choo, et al., Science 244: 359-362 (1989)), hepatitis C virus (HCV) is now widely accepted as the most common causative agent of post-transfusion non A, non-B hepatitis (NANBH) (Kuo, et al., Science 244:362-364 (1989)). Infection with HCV is a major cause of human liver disease throughout the world with seroprevalence in the general population ranging from 0.3 to 2.2% (van der Poel, et al., HEPATITIS C VIRUS; Amsterdam: Karger; pp. 137-163 (1994)) to as high as .about.10-20% in Egypt (Hibbs, et al., J. Infect. Dis. 168: 789-790 (1993)). Although the virus is most commonly transmitted via blood, the mode of transmission remains unknown for a large portion of infected individuals (Alter, M. J., Infect. Agents Dis. 2: 155-166 (1993)). Over 50% of infections by HCV progress to acute hepatitis associated with viremia and generally elevated serum alanine aminotranferase (ALT) levels (Alter, H. J., CURRENT PROSPECTIVES IN HEPATOLOGY; New York: Plenum; pp. 83-97 (1989)). Over half of the acute cases progress to a chronic infection with roughly 20% developing cirrhosis (Alter, H. J., supra). Chronic infection by HCV has also been linked epidemiologically to the development of hepatocellular carcinoma (HCC), especially in cirrhotic patients (Blum, et al., Hepatology 19: 251-258 (1994)).
Since its initial identification, many isolates of HCV have been sequenced, displaying much genetic diversity and leading to the subclassification of this virus into multiple genotypes. See, e.g., Bukh, et al., Proc. Natl. Acad. Sci. USA 90:8234-8238 (1993); Bukh, et al., Proc. Natl. Acad. Sci. USA 91: 8239-8243 (1994); Dusheiko, et al., Hepatology 19: 13-18 (1994)). Due to its genome structure and sequence homology, this virus was assigned as a new genus in the Flaviviridae family, along with the other two genera, flaviviruses (such as yellow fever virus and Dengue virus types 1-4) and pestiviruses (such as bovine viral diarrhea virus). See, e.g., Choo, et al., supra; Miller, et al., Proc. Natl. Acad. Sci. USA 87: 2057-2061 (1990)). Like the other members of the Flaviviridae family, HCV is an enveloped virus containing a single-stranded RNA molecule of positive polarity. The HCV genome is approximately 9.6 kilobases (kb) with a long, highly conserved, noncapped 5' nontranslated region (NTR) of approximately 340 bases which functions as an internal ribosome entry site (IRES) (Wang, et al., Curr. Topics Microbiol. Immunol. 203: 99-112 (1995)). This element is followed by a region which encodes a single long open reading frame (ORF) encoding a polypeptide of .about.3000 amino acids comprising both the structural and nonstructural viral proteins. This large polypeptide is subsequently processed into the individual structural and nonstructural proteins by a combination of host and virally-encoded proteinases (reviewed in Rice, VIROLOGY; Raven Press: New York, 2nd Ed.; pp. 931-960 (1996)). In their respective order in the polyprotein precursor from amino-terminus to carboxy-terminus, are core-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B.
Inhibition of the biological activity of viral proteins of the Flaviviridae family, particularly HCV NS5B, is potentially of benefit in controlling, reducing and alleviating the diseases caused by infection with these viral organisms. Clearly, there is a need for factors, such as the novel compounds of the invention, that have a present benefit of being useful to screen compounds for antiviral activity. Such factors are also useful in determining their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.
In an attempt to identify the critical domains of the HCV NS5B protein, Lohmann, et al., J. Virol. 8416-8428 (1997) truncated both the amino and carboxy termini of the full-length HCV NS5B protein. While Lohmann, et al. discuss various sequence fragments of the HCV NS5B protein, with one exception of a known GenBank sequence, the reference does not specifically disclose either the cDNA or amino acid sequences of the these fragments.
Clearly, there is a need to discover new antiviral compounds that are useful in the cure, treatment, and prevention of virus infection of the Flaviviridae family, particularly HCV. Because the HCV NS5B protein is difficult to express, it is important to discover forms of the protein which may be more soluble for use in a drug-discovery screen than is the full-length HCV NS5B protein. The instant invention is believed to provide for such a need in the antiviral area.